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| United States Patent |
5,149,629
|
|
Rishpon
, et al.
|
September 22, 1992
|
Coulometric assay system
Abstract
The invention relates to a novel assay for the determination of entities
having biological activity and to a device for carrying out such
determinations. The assay is a very sensitive one and quantities in the
picogram per milliliter range can be determined. The assay is based on
chrono-coulometric measurements with sequential measurements of a
plurality of samples being carried out with the aid of a multiplexer, in
combination with a potentiostat, there being provided suitable electrodes
and means for applying a predetermined voltage. One of the electrodes is
advantageously a glassy carbon electrode, carbon felt or cloth or carbon
paper. Amongst biologically active entities there are antibodies/antigens;
hormones/receptors; nuclotides/nucleotide probes, one of the members of
such a pair being immobilized on an electrode surface, being tagged with
an enzyme providing a signal in coulometric measurements.
| Inventors:
|
Rishpon; Judith (Wolfson, IL);
Rosen; Ilara (Ramat-Gan, IL)
|
| Assignee:
|
Ramot University Authority for Applied Research and Industrial (Tel Aviv, IL)
|
| Appl. No.:
|
177463 |
| Filed:
|
April 4, 1988 |
Foreign Application Priority Data
| Current U.S. Class: |
435/7.9; 435/7.5; 435/287.2; 435/287.9; 435/817; 436/527; 436/806 |
| Intern'l Class: |
G01N 033/53; G01N 033/552; C12M 001/40 |
| Field of Search: |
204/1 T,403
436/525,527,806
435/7,18,21,25,26,288,291,817,7.9,7.5
|
References Cited
U.S. Patent Documents
| 4228237 | Oct., 1980 | Hevey et al. | 435/7.
|
| 4619754 | Oct., 1986 | Niki et al. | 204/403.
|
| 4711245 | Dec., 1987 | Higgins et al. | 204/403.
|
| 4713165 | Dec., 1987 | Cononer et al. | 204/403.
|
| Foreign Patent Documents |
| 0170375 | Feb., 1986 | EP.
| |
Other References
Robinson, G. A., et al., Biological Abstracts, vol. 82, Abstract No. 91756
(1986).
Strohl, A. N., et al., Chemical Abstracts, vol. 91, No. 1, Abstract No.
2042t (1979).
|
Primary Examiner: Kepplinger; Esther L.
Assistant Examiner: Scheiner; Toni R.
Attorney, Agent or Firm: Browdy and Neimark
Claims
We claim:
1. Apparatus for the sequential rapid qualitative or quantitative assay of
a plurality of samples of members of biospecific binding pairs by
coulometric measurement, comprising:
a plurality of working electrodes corresponding to the number of samples to
be assayed, said electrodes immersed in a common solution in a vessel,
a multiplexer for effecting sequential coulometric measurements,
a potentiostat;
there being provided in said vessel a reference electrode and a counter
electrode, means for applying a predetermined voltage during each such
measurement;
which measurements are that of the electric charge passing between each
working electrode and the evaluation thereof indicating the quantity of
one of the members of the biospecific binding pairs, or its presence, said
working electrodes comprising an electrically inert support which supports
a member selected from the group consisting of carbon felt, carbon paper
or carbon cloth to which there is firmly bonded one of the biospecific
pair members which specifically binds the second complementary member,
which second member is tagged by an enzyme or which second member is
coupled to at least one further biospecific member, the last of which
further biospecific members is tagged with an enzyme;
whereby when the electrodes are introduced into a substrate of the enzyme,
a coulometric signal is formed, thereby sequentially measuring said
samples.
2. Apparatus according to claim 1 wherein each working electrode comprises
a carbon felt attached to an electrically non-conducting inert support,
said felt being electrically connected to the multiplexer.
3. Apparatus according to claim 1 wherein one of the members of the
biospecific binding pairs is bonded to the carbon felt, paper or cloth of
the working electrode by a carbodiimide linkage.
4. An apparatus according to claim 1 further comprising means for rotating
the working electrodes during the incubation of the two members of the
binding pair or during the time of measurement.
5. An apparatus according to claim 1 wherein means are provided for
connecting and disconnecting at will the potential applied to the working
electrodes between measurements.
6. An assay for the qualitative or quantitative sequential determination of
a plurality of samples of members of specific binding pairs selected from
the group consisting of antibodies/antigens, hormones, receptors, and
nucleotides/nucleotide probes, wherein a first member of a pair is
immobilized in an apparatus as claimed in claim 1, wherein the method
comprises:
(a) contacting the working electrode with a sample containing the second
member of the pair, said second member being labelled with an enzyme, so
as to bind said second member to the first member immobilized on the
electrode,
(b) immersing the electrode into a solution containing a substrate of the
enzyme,
(c) generating a coulometric signal,
(d) measuring and evaluating said signal, and
(e) repeating said assay sequentially for each sample.
7. A sequential assay according to claim 6 wherein a known amount of
biotin-tagged antigen is present together with an unknown amount of the
same antigen in an aqueous system, wherein an antibody is bound to the
working electrode, and where the antibody bound to the electrode reacts
with a biotin-labelled antigen and an unlabeled antigen followed by
incubation with an avidin-conjugated enzyme, wherein one of the members of
the biotin-tagged antigen and the avidin-conjugated enzyme is bonded to a
carbon felt, carbon paper or carbon cloth by a carbodiimide linkage.
8. An assay according to claim 6 wherein the linkage of the one member of
the biospecific binding pair to the carbon felt of the working electrode
is via a linkage selected from the group consisting of carbodiimide or
biotin-avidin linkage.
9. An assay according to claim 6 wherein the working electrodes are rotated
during the period of incubation of the binding pair and during the time of
the actual coulometric measurement.
10. An assay according to claim 6 wherein the working electrodes are
rotated during the period of incubation and during the period of time of
actual coulometric measurement.
11. An assay according to claim 6 wherein means are provided for
disconnecting any of the working electrodes during the period between
actual measurement, and reconnecting said electrodes to the source of
electric potential during the measurement.
Description
FIELD OF THE INVENTION
The invention relates to a novel assay for the determination of
biomolecules by electrochemical means. The invention relates to such
quantitative determinations based on the coupling of certain types of
biomolecules to be determined to enzymes and to the use of electrochemical
enzyme sensors bearing such enzymes. The invention further relates to a
device for the rapid sequential determination of large numbers of samples,
which is of considerable utility in large-scale determinations such as in
clinics or hospitals.
BACKGROUND OF THE INVENTION
A wide variety of tests is used for the detection and for the quantitation
of biomolecules. A high sensitivity is required for many of these as they
are present in biological fluids in very small concentration. Furthermore,
the assays must have a high degree of selectivity in order to be able to
determine a specific biomolecule in the presence of other entities.
Many tests are based on the coupling of the species to be determined to
another moiety and the subsequent determination of the labeled entity.
There exist various assays based on the interaction of enzymes and their
substrates; on the interaction of antigens and antibodies, between
hormones and receptors, etc. There exists a wide variety of assays which
are based on radioactive labels and on other types of labels.
The translation of selective interaction into a measurable quantity often
implies coupling of a detectable labeling to one or more of the
interacting species. Radioisotope labeling is one kind of such labeling
which is used in analytical clinical laboratory.
However, the desire to avoid the use of radioactive techniques has
stimulated the development of other labels. Among these enzymes appear to
be practical: an enzyme is coupled with the bioactive material as a marker
and the enzyme activity is measured. Enzyme labels increase the
sensitivity through chemical amplification. Chemical amplification refers
to the passing of a substance through a catalytic cycling, or
multiplication mechanism to generate a relatively large amount of product.
The rate at which the product is formed is related to the concentration of
the analyte in the sample. The enzyme activity is usually measured by
optical instruments such as colorimeters or spectrophotometers. The
development of electrochemical bioassays has receive only little
attention. Electrochemical methods are free of sample turbidity quenching,
and interferences from the many absorbing and fluorescing compounds in
typical biological samples that hinder spectroscopic techniques. Enzyme
electrodes are an example of the combination of enzyme action with
electrochemical measurements for analytical purposes. Enzyme electrodes
are a type of biosensors. Biosensors are analytical devices in which
biological materials capable of specific chemical recognition, are in
intimate contact with transducers. Among these, bioelectrochemical sensors
such as enzyme electrodes have found promising application especially in
clinical and process measurements. Commercial analyzers equipped with
enzyme sensors are available mostly for serum components measurements.
There have been several attempts to construct other biosensors such as
e.g. immunosensors.
However, at present amongst the disadvantages of such sensors are the
following: The sensitivity is low as compared with established methods
such as enzyme immuno-assays and a measuring cell is occupied for the
whole incubation time to form an antigen-antibody complex. Therefore the
advantages of electrochemical sensors-their short response time-is not
exploited efficiently. Only a few samples can be measured per day and fast
measurements are impossible. This is true especially in clinical tests
which usually involve a large number of samples. The use of a single
electrode and the calibration it requires prior to measurements impedes
the measurements and does not allow its successful application in clinical
tests in hospitals and clinics.
The system can be used to carry out assays with a wide variety of
biointeractions. The invention is illustrated in the following with
reference to a number of specific examples, which are to be construed in a
non-limitative manner.
One of the representative enzymes which provide for a wide scope of
substrates is alkaline phosphatase.
The enzyme alkaline phosphatase enzyme is a common label in immunological
tests. Conjugates of antigens and antibodies with this enzyme are
commercially available in a rather purified form. The common substrates
for alkaline phosphatase used in various immunological tests are
nitrophenyl phosphate and phenylphosphate. Electrochemical determinations
of alkaline phosphatase based on the hydrolysis products of the enzyme
reaction are rather difficult because of the high over-voltage of the
phenol compounds and serious problems arising from adsorption to the
electrode and fouling of the electrode response.
SUMMARY OF THE INVENTION
According to the present invention there is provided a convenient and rapid
assay for the quantitative determination of a wide variety of molecules
having a biological activity.
The invention further provides a device for the rapid and convenient
chronocoulometric determination of such entities. There is provided an
automated multi-sample and multi-electrode system for carrying out such
determinations. There is further provided a convenient method of tagging
of molecules to be determined, wherein alkaline phosphatase is used in
conjunction with p-aminophenyl phosphate. Amongst entities which can be
thus determined with a high degree of accuracy and very high sensitivity
there may be mentioned antigens, antibodies, hormones, etc.
The multi-electrode system of the invention makes possible a rapid assay of
a plurality of samples and is of special use in applications such as
clinical and hospital use where a plurality of samples has to be analysed
within as short a period of time as possible. The device comprises a
plurality of enzyme electrodes, a counterelectrode and a reference
electrode, there being provided means for rapidly switching over from one
such electrode to another there further being provided a computerized
system for the rapid recordal and evaluation of the results.
The basis of the measurement is based on the application of a predetermined
voltage between the reference and the working electrode (say of the order
of 0.zV for a certain enzyme); and measuring the electrical current at
such voltage for each of the electrodes.
There are preferably provided means for the rotation of the electrodes as
the measurement is being carried out. Each of the electrodes is first
inserted into a different sample and incubated therein, and after this
there is carried out the measurement in the device wherein there is
provided a multiplexer, such sequential measurement in the same vessel
being possible as the concentration of the solution does not undergo any
appreciable change during such measurement. It is clear that the system
illustrated herein, with 8 enzyme electrodes, is by way of example only
and that there can be provided a larger number of such electrodes with
suitable multiplexing and auxiliary equipment. For example, if an enzyme
is to be determined which is an antigen, there can be bonded to a suitable
electrically conducting electrode antibodies specific for such enzyme; the
electrode is contacted with the sample containing the antigen, and if this
is an enzyme which provides a product of reaction which reacts at the
electrode, the electrode is introduced into an excess of substrate and the
current is measured at a constant voltage, being indicative of the
quantity of antigen. The measurement is always carried out when there is
no saturation respective the antibodies. t is possible to use competitive
methods of measurement where a given concentration of the tagged entity
makes possible the determination of the untagged one by the determination
of the ratio of these, measurements being made with various concentrations
of the tagged moiety. The attachment of the desired entities (in this case
antibodies) can be effected by chemical bonding or by mere adsorption.
Details of the techniques used are set out in the experimental part.
Experiments were carried out with IgG (anti-mouse IgG) which is a
representative sample of any antigen and with dog IgG which is
representative of an antigen-antibody system. Amongst hormones there may
be mentioned the determination of aldosterone; HCG etc. which are
representative of a wide variety of hormones. There may be attached to the
electrode polyclonal or monoclonal antibodies, such as anti-IgG. It is
possible to produce antibodies specific against certain hormones and to
attach such antibodies to the electrode: for example insulin, T.sub.3,
T.sub.4, etc., where the antibody has the function to serve as carrier
making possible the reaction at the electrode set out above. The invention
is illustrated with reference to the enclosed FIGS. in which:
FIG. 1 is a schematic block diagram of a device of the invention;
FIG. 2 illustrates the response of an mIgG electrode to the addition of
amino-phenyl phosphate, with IgG concentrations indicated on the abscisa;
FIG. 3 illustrates the measurement of Dog IgG on an electrode of the
invention, the measurement being with dog serum on the electrode;
FIG. 4 illustrates the detection of .beta.-HCG in urine;
FIG. 5, illustrates the electrode response as a function of HCG
concentration.
FIG. 6 illustrates the determination of .beta.-HCG using carbon felts as
support (electrode);
FIG. 7 illustrates the system of MIG-Biotin-Carbon felts;
FIG. 8 illustrates the system of Biotin-MIG-carbon felts.
The device of the invention is schematically illustrated herein with
reference to FIG. 1.
FIG. 2 illustrates the use of such system with an antimouse-IgG electrode,
showing the effect of the addition of amino phenyl phosphate and the
response of an mIgG electrode to such addition, with IgG concentration
noted on the abscissa.
FIG. 3 illustrates the determination of dog IgG: Different dog serums
(diluted 1:10 in phosphate buffer pH 7.5 0.2M) were dried in the presence
of carbodiimide on gold electrodes. The electrodes were then allowed to
react with antidog IgG conjugate to alkaline phosphatase. The electrode
signal was proportional to the IgG level in the dog serum that was on the
electrode. The results are summarized in FIG. 3. The bars represent the
electrode response to phenyl phosphate after incubation with different
concentrations of antidog IgG alkaline phosphatase conjugates. The
immunoglobulin concentration in the serum:
______________________________________
Left bar blank, no serum
Middle bars serum IgG level 3 mg/ml
Right bars serum IgG level 6 mg/ml.
______________________________________
Measurements were done in the presence of 10 mM MgCl.sub.2 and the solution
was stirred by magnetic stirrer.
In FIG. 4: illustrates the detection of .beta.- HCG in urine.
Left bars--response of gc/aHCG (polyclonal) electrode to aminophenyl
phosphate (2 mM) after incubation with urine samples containing different
concentrations of HCG (1 hr, 37.degree. C.) followed by ap-aHCG
(monoclonal) (1 hr, 37.degree. C.) (sandwich).
Right bars--Response of gc/a.beta.-HCG (monoclonal) electrode to
aminophenyl phosphate (2 mM) after incubation with urine samples followed
by a.beta.HCG (rb) (policlonal) and ap-arb IgG (1 hr, 37.degree. C.)
(double sandwich).
FIG. 5 illustrates the determination of .beta.-HCG in serum. Electrode
response as a function of .beta.HCG concentration: Serum samples in which
the HCG concentration was determined by radio immunoassay were diluted in
PBS and incubated with electrodes on which monoclonal antiHCG was
immobilized. The device for carrying out the assay with a plurality of
samples in rapid sequence is illustrated with reference to FIG. 1, where
this is illustrated in a schematical manner. The device comprises a vessel
11, shown in a view from above, wherein there are positioned a number of
identical electrodes 12, 13 and 14, 15, 16, 17, 18 and 19, each of which
is made of an electrically conducting inert material (such as carbon,
graphite, gold-plated or platinum plated metal), in a rod shape, embedded
in a non-conducting inert material such as teflon. Advantageously, each of
the electrodes 12 to 19 is provided for means of axial rotation, as
indicated by the arrows 20 and 21. Such rotation substantially decreases
the period of time required for the biological interactions and thus
decreases the time of measurement. The rotation also increases the
electrode signal.
The system comprises a microcomputer 22, connected with a potentiostat 23,
the connection being via a digital to analog port. The electrode assembly
is connected to the potentiostat 23 and also via the multiplexer 24 to a
parallel port 25 of the microcomputer 22. The electrodes 12 to 19 are all
in the vessel 11 which contains the reference electrode 26 and the
counterelectrode 27, which are also connected to the potentiostat 23. The
mode of operation is amperometric, i.e. a potential is applied to the
working electrode and the resulting current is measured. Each of the
electrodes can be connected and disconnected at will, and thus by hopping
from electrode to electrode, a plurality of measurements can be effected
within a brief period of time. While such measurements are effected, the
electrode in operation is rotated.
An enzyme electrode operated in this mode has an increased response. At the
time interval when the electrode is disconnected the product of the
enzymatic reaction is accumulated near the electrode, a larger amount of
the product of the enzymatic reaction reacts at the electrode compared
with such electrode connected continuously, and the current is higher.
A higher sensitivity is obtained by the integration of the current signal.
The integration is effected digitally by the computer which considerably
increases the signal to noise ratio. Such integration starts a few
milliseconds after application of the potential on the electrode at each
cycle and thus interference from double layer charging and other surface
reactions are suppressed.
The system of the invention can be used for quantitative measurements of a
high degree of sensitivity, with sensitivities in the picogram/ml range.
The system can also be used for screening procedures (such as for example
for the presence or absence of breast cancer and other malignancies).
The assays can be based on a variety of enzymes, one of the enzymes of
choice being alkaline phosphatase. Other enzymes which can be used are of
the hydrolase type, such as aryl galactosidase; dehydrogenases such as
alcohol dehydrogenase, glucose dehydrogenase, etc.; oxidases such as
glucose oxidase, alcohol oxidase, etc. The systems can be based on
antigen-antibody interaction as well as systems such as hormone-receptor;
nucleoside in one strand of a DNA or RNA and the nucleotides in a
complementary strand, etc.
In general terms, the analytical system of the invention can be used with
systems in which one type of molecule (A) binds specifically with a high
binding constant with a second type (B), said binding molecules (A) being
capable of being immobilized on a solid inert electrically conductive
surface such as glassy carbon, graphite, gold, etc., such bound (A)
molecules being able to interact and bind to said (B) type molecules which
will be specifically adsorbed on the electrode surface.
According to one embodiment of the invention, the electrochemical assay can
be carried out with alkaline phosphatase. This is effected with such
alkaline phosphatase attached to an electrode, where the substrate is
p-aminophenyl phosphate. This substrate was prepared for this purpose from
nitrophenyl phosphate, by catalytic hydrogen reduction of the nitro group
in the presence of platinum oxide. The thus obtained aminophenyl phosphate
is perfect for use with the above described sensor. The assay is effected
as set out above, and the product of the enzymatic hydrolysis is
amino-phenol which is easily oxidized at a carbon electrode: the
electrochemical determination of alkaline phosphatase using this substrate
occurs at a low overvoltage of the order of about 0.2 V versus SCE, and
the electrode reaction is free of phenomena such as adsorption or fouling.
The invention is illustrated in the following with reference to antigens
and antibodies. It can be used for the quantitative assay of corresponding
systems such as hormones, receptors, nucleotides of complementary nucleic
acids and the like. The assay can be based on competition reactions, on
sandwich type reactions, etc.
Representative stages of an assay according to the invention are:
A. Equal quantities of antibodies are immobilized on each one of the
electrodes (by covalent binding or simply by adsorption).
B. In a competition assay, each electrode is allowed to react with a
solution in a test tube which contains the antigen, the concentration of
which is to be measured, together with a known amount of the antigen
conjugated to an enzyme.
In the sandwich-type assay, each electrode is allowed to react with a
solution containing the antigen, the concentration of which is to be
measured.
C. All the electrodes are washed in a solution containing a detergent and
in some cases also bovine serum albumin.
In the competition procedure the assay is continued from stage E, and stage
D is skipped.
D. In the sandwich procedure the electrodes are inserted into a beaker
containing a solution of a second antibody conjugated to an enzyme (e.g.
alkaline phosphatase) and allowed to interact with this solution. The
electrodes are then washed with a detergent solution.
E. The whole electrode assembly is inserted in an electrochemical cell
containing a buffer solution which is optimal for the enzyme action and
the electrodes are connected to a potentiostat which is connected to the
microcomputer. Each electrode is also connected to an electronic switch
which is connected to a parallel port of the computer.
The chrono-coulometric mode is then applied for the detection of the enzyme
amount on each one of the electrodes. In the beginning of the
electrochemical measurement all the electrodes are operated together,
connected and disconnected repeatedly, which shortens the time of their
equilibration. After this the computer scans all the electrodes via the
parallel port, and the background response to the potential application of
each electrode is recorded by the computer. The substrate for the enzyme
is then added to the cell and the response of each electrode is recorded
and integrated by the computer. The whole electrochemical measurement
sequence can be completed in about two minutes after the addition of the
substrate. The response of each electrode is related to the amount of
enzyme on that electrode.
The electrochemical measurements do not affect the immunological
interactions, and thus it is possible to continue the last incubation
stage after the electrochemical stage.
The electrodes can be washed and then used to react with the solutions
containing the alkaline phosphatase conjugates for a longer period of time
and then the electrochemical assay can be repeated. This is impossible
with immunoassays using the ELISA technique.
F. In cases where the electrodes surfaces are not identical, the system can
be calibrated by measuring the oxidation or reduction of an electroactive
species such as oxidation of ferrocyanide in the electrochemical cell and
comparison of the results of all the electrodes.
An enzyme of choice for use in the assay of the invention, is alkaline
phosphatase attached to an electrode in which the substrate is
p-aminophenyl phosphate. This substrate was synthesized from nitrophenyl
phosphate. The synthesis was carried out by catalytic hydrogen reduction
of the nitro group in the presence of platinum oxide (1). The product of
the enzymatic hydrolysis is amino phenol which is easily oxidized at an
inert electrode such as a carbon electrode. Thus the electrochemical
determination of alkaline phosphatase using this substrate occurs at low
over voltage (.about.0.2 volts vs SCE) and the electrode reaction is free
of problems such as adsorption and fouling. The following examples are to
be construed in a non-limitative manner.
EXAMPLES
A. Anti-mouse IgG (200 nanogram) were covalently bound by the carbodiimide
method (3) to carbon electrodes of 0.07 cm.sup.2 area (glassy carbon or
graphite). The electrodes were then allowed to react with 0.25 ml of mouse
IgG (24 microgram/ml) for 1 hour and then with a second antibody directed
against mouse IgG and conjugated to alkaline phosphatase (Bio-Yeda catalog
No. 3465-1) diluted 1:500 again for 1 hour. The amount of IgG in the test
samples could be calculated from the electrode response.
B. Antibodies against aldosterone were bound to the electrodes and the
electrodes were allowed to react for 30 minutes with test samples
containing unknown amounts of aldosterone and known amount of aldosterone
conjugated to alkaline phosphatase. The aldosterone concentration in the
test sample can be measured with a sensitivity in the picogram/ml range.
MULTIELECTRODE SYSTEM
Clinical tests in hospital and clinical laboratories usually involve a
large number of samples. In addition, measurements based on biologically
active materials such as enzymes and antibodies do need calibration curves
and standards. Hence a system capable of simultaneous measurements will be
preferred.
A multielectrode system which allows the simultaneous determination of
different samples has been designed. An instrument composed of eight
electrodes has been built and successfully tested.
The instrument is composed of an ensemble of identical electrodes made of
glassy carbon in a rod shape embedded in teflon. The electrodes assembly
is connected to the potentiostat and is also connected via a multiplexer
to a parallel port of the microcomputer. The electrodes are all inserted
in an electrochemical cell containing a reference electrode and a counter
electrode which are also connected to the potentiostat. Each electrode can
be electrically disconnected for some time interval and then connected
again in a repeated mode. An enzyme electrode operating in this mode has
increased response. At the time interval when the electrode is
disconnected the product of the enzymatic reaction is accumulated near the
electrode. Thus, when a potential is applied for a short time interval on
the electrode, a larger amount of the product of the enzymatic reaction
reacts at the electrode than if the electrode is connected continuously
and the current is higher. In addition, higher sensitivity is obtained by
integration of the current signal. The integration which is done digitally
by the computer considerably increases the signal to noise ratio. The
integration starts few milliseconds after application of the potential on
the electrode at each cycle and thus interference from double layer
charging and other surface reactions is supressed. The time when the
electrode is disconnected is used for the detection of other electrodes.
Thus, with a computer controlled electronic switching one can measure many
electrodes in the same solution. Since the enzyme is confined only to the
electrode surface there are no interferences between the electrodes.
The instrument built enables the simultaneous determination of samples on
eight electrodes. Similar devices containing a larger number of electrodes
can be prepared. It considerably reduces the time needed for the
electrochemical assay. In addition, since all the electrodes are tested in
the same solution and their response to the addition of the same amount of
substrate is checked, errors introduced by dilutions etc. are minimized.
This multielectrode system was tested using the mode system mouse
immunoglobulins and also the hormone .beta.HCG.
DISPOSABLE ELECTRODES
Rather then devise a reusable electrode in which a recovery procedure is
required, we, recently, worked out a procedure employing very inexpensive
disposable electrode systems.
Three types of carbon electrodes were tested: carbon paper, carbon cloth
and carbon felts. Among the three, the carbon felt electrodes have the
highest reproducibility. The carbon felt was cut into small discs
(diameter--4 mm and height--0.6 mm).
The immunoassays were carried out using the model system mouse IgG
Antimouse IgG (polyclonal) were covalently coupled to carbon felt discs by
the use of EDC. The discs were left overnight in a solution containing the
antibodies and EDC (10:1 w/w). The discs were then washed and allowed to
react with a solution containing mouse IgG (diluted 1:1000) for 1 hour,
and then with a solution of antimouse IgG conjugated to alkaline
phosphatase again for 1 hour. At this stage each disc was mounted on a
teflon rod to which a platinum wire was inserted for electrical
connection. The discs were then checked electrochemically for alkaline
phosphatase activity using the same assay described for the glassy carbon
electrodes. Table III summarizes the results obtained with the carbon felt
electrodes and mouse IgG system.
DETERMINATION OF THE HORMONE .beta.HCG.
Two procedures have been developed and tested for the hormone .beta.HCG.
A. The sandwich procdure: Ponyclonal antibodies against HCG (aHCG) were
covalently bound to glassy carbon electrodes. The electrodes were then
allowed to react with test samples containing unknown amount of the
hormone for 1 hour, and then washed and transferred to a solution
containing monoclonal antibodies against HCG conjugated to alkaline
phosphatase (ap-a .beta.HCG)and incubated for 1 hour. From the
electrochemical assay which followed, the hormone concentration could be
determined.
B. The double sandwich procedure: Monoclonal antibodies against .beta.HCG
were attached to glassy carbon electrodes by covalent binding or even by
simple adsorption. The electrodes were then allowed to react with test
samples containing unknown amount of the hormone for 1 hour at 37.degree.
C. and then washed and transferred to a beaker containing polyclonal
antibodies against HCG which were produced in a rabbit, and incubated at
37.degree. for 1 hour. The electrodes were then transferred to a solution
containing antibody against rabbit immunoglobulins conjugated to alkaline
phosphatase, (ap-arb IgG) and then washed and tested electrochemically for
alkaline phosphatase activity. From the electrochemical assay the hormone
concentration could be determined. FIG. 4 shows results obtained by the
determination of .beta.HCG in urine samples. From FIG. 9 it is obvious
that the double sandwich procedure described in procedure B is more
sensitive. It involves an additional incubation step compare to procedure
A, but on the other hand the alkaline phosphatase conjugates used are less
expensive and are commercially available. Hence, at the next stages we
focused our attention in standardization and optimization of the the
.beta.HCG assay according to procedure B.
The following results were obtained using monoclonal and polyclonal
antibodies against HCG purchased from Bio-Makor, Israel, and antirabbit
conjugates purchased from Sima, U.S.A. Table I summarizes a a set of
experiments in which the hormone was measured in PBS solutions.
TABLE I
______________________________________
Determination of .beta.HCG in buffer solutions
.beta.HCG U/L
0 10 20 40 80 160 320
______________________________________
Q uC 2.03 2.75 2.45 4.86 7.91 8.57 17.0
n 12 17 8 20 16 13 1
SD 0.41 0.65 1.27 2.72 5.54 4.3 --
______________________________________
.beta.HCG U/L = the concentration of HCG
Q = Average electrode response in microcoulomb/10 seconds
n = no of electrodes tested at each concentration
SD = standard deviation at each concentration
In another set of experiments serum samples containing a known amount of
.beta.HCG (determined by the radioimmunoassay) were diluted in PBS and
measured for the hormone (FIG. 5)
Table II summarizes a set of experiments in which the hormone HCG was added
to serum samples diluted 1:5 in PBS. These serum samples were tested for
the hormone by the radio immunoassay technique and were found negative.
TABLE II
______________________________________
Determination of .beta.HCG added to serums
.beta.HCG U/L
0 40 80 160
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Q uC 1.57 3.00 4.62 5.47
n 3 2 10 3
SD 0.21 0.60 1.77 1.27
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In parallel to the elelctrochemical determination we routinely measured the
reagents used by application the standart ELISA technique. In general, the
results obtained by the ELISA technique showed a high background and the
lowest detection limit was much higher compared with those obtained by the
electrochemical measurement.
TABLE III
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Mouse IgG measured by carbon felt discs
(Each electrode checked separately)
Measurements were carried out in duplicates (I and II)
amIgG IgG amIgG Q I Q II Ave
ug/ml ug/ml (dilution)
(uC) (uC) u(uC)
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0.5 10 1:500 47.5 40 43.7
" " 1:1000.vertline.
43 30 36.5
" -- 1:500 10.9 13 11.9
" -- 1:1000 4 4.2 4.1
20 10 1:500 157 109 133
" " 1:1000 63.6 60 61.8
" -- 1:500 19 20 19.5
" -- 1:1000 7 5.2 6.1
______________________________________
These results demonstrate that the sensitivity attainable with the carbon
felts is even higher then that of the glassy carbon.
The carbon felts were also integrated into the multielectrode system.
Results are summarized in Table IV.
TABLE IV
______________________________________
amIgG IgG amIgG-A Q I Q II Ave
ug/ml ug/ml (dilution)
(uC) (uC) u(uC)
______________________________________
0.5 10 1:500 124 86 105
" " 1:1000 60 85 72.5
" -- 1:500 17.8 12 14.9
" -- 1:1000 10.8 10.9 10.9
______________________________________
Measurements were taken in duplicates (I and II)
Biotin Labeled Antigens
The highly specific and strong binding of biotin to avidin was utilized for
the determination of mouse IgG. Biotin can also be attached to sugars,
DNA, etc. and hence the method described can be used for the determination
of biological interactions in general.
Mouse antibodies were labeled by biotin by the N-hydroxy succinimide method
(Bayer E.A. and Wilchek M, 1980, in "Methods of Biochemical Analysis", 26,
1-45). Extra Avidin Alkaline phosphatase conjugates were purchased from
Biomakor. Carbon felt Type RVG 1000 purchased from Carbon Lorraine France
was used for preparation of the disposable electrodes. The felt was cut
into disk 4 mm diameter and 0.6 mm thick. Antimouse IgG were immobilized
on the carbon by the carbodiimide method. The carbon felt disks were left
overnight in a solution containing 20 microgram/ml antimouse IgG and 2
microgram/ml EDC at room temperature. The disks were then washed in Tris
buffer and transferred into Elisa plates (a carbon disk per well) and
incubated with mouse IgG solution for one hour at different
concentrations. The disks were then washed and incubated with solution
containing extra avidin conjugated to alkaline phophatase. After washing
the disks were mounted on a teflon holder and attached to platinum wire
for electrical; connection and were then tested for alkaline phophatase
activity. The alkaline phosphatase activity was related to the mouse IgG
concentration. Results are summarized in FIG. 7 and FIG. 8.
F. A droplet (5 microliters) of a solution containing monoclonal antibodies
against viral antigen associated with breast cancer were dried on glassy
carbon electrodes. The electrode were then allowed to react for 1 hour at
37.degree. with sera (diluted by 10) or plural fluid (diluted up to 500)
of patient suffering from breast cancer and patients that do not have the
disease. The electrodes were then allowed to react with alkaline
phosphatase conjugates to monoclonal antibodies against the viral antigen
and for 1 hour at 37.degree. or for 16 hours at room temperature then
tested electrochemically. The electrochemical measurement showed the
existence of the antigen in the patients suffering from the disease. This
provides a sensitive test for determining the presence or absence of
breast cancer. Other examples that are currently tested are the detections
of other hormones such as T3 and T4 and insulin levels (in blood or
urine).
REFERENCES:
1. J. C. Moffart and H. G. Khorana, J. Am.Chem.Soc. 79, 3741, (1957)
2. F. W. Scheller, F. Schubert, R. Renneberg, H. G. Muller, M. Janchen & H.
Weise, Biosensors, 1, 135, 1985.
3. Laval, Bourdillon & Moiroux, J. of Amer. Chem. Soc. 106 4701 (1984).
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